Research Advances Role of STIM2 in SOCE and Regulation of Cell Function: Previously we demonstrated the role of STIM2 in regulating SOCE and cell function. We showed that STIM2 facilitates (i) STIM1 clustering and (ii) STIM1-gating of Orai1 at relatively high ER-Ca2+, i.e. conditions that likely do not elicit a STIM1 response; (iii) remodeling STIM1 C-terminus into the active conformation. Our key findings in this fiscal year are as follows: 1. Key role for STIM2 in regulation of NFAT1 signaling: Activation of nuclear factor of activated T-cells (NFAT) 1-dependent gene expression in the nucleus is a vital cell function that is regulated by SOCE. Defects in this pathway or aberrant function of either Orai1 or STIM1 proteins are associated with immunodeficiency and autoimmunity in patients and mouse models. STIM1 is the primary activator of Orai1 as loss of the protein eliminates Orai1 activation and consequently, regulation of downstream Ca2+-dependent cell functions. STIM2 is reported to induce relatively weak activation of the channel. Intriguingly, although STIM2 is not a major contributor to SOCE in most cell types, it appears to have a unique physiological relevance. Mice lacking the protein displayed developmental defects and died 4-6 weeks after birth. Further, knockdown of the protein in T-cells caused small decrease in SOCE, in contrast to STIM1-deficient T cells that had almost complete loss of SOCE. Importantly, STIM2-deficient cells display significantly greater decrease in cytokine production due to a defect in nuclear translocation of NFAT1 (15). Together, these previous findings suggest a specific role for STIM2 in SOCE-dependent NFAT activation. We have examined the contribution of STIM2 to NFAT1 activation in response to Ca2+ entry via Orai1/STIM1 channel. Our findings demonstrate that while clustering of STIM1 with Orai1 in ER-PM junctions induces sufficient increase in both local and global Ca2+i to trigger NFAT1 activation, assembly of Orai1/STIM1 with STIM2 is essential for targeting the channel to specific ER-PM junctions which are permissive for assembly of Orai1 with the AKAP79 signaling complex. Interaction of Orai1 with AKAP79 is an essential step in coupling Orai1 function with NFAT1 activation since it allows local Ca2+ increases mediated by the channel to be utilized for activating calcineurin that is scaffolded by AKAP79. In aggregate, our findings establish a critical, and essential, role for STIM2 in targeting Orai1/STIM1 to specific ER-PM junctions that permit assembly of the channel with AKAP79 signaling complex. Compartmentalization of Orai1/STIM1 by STIM2 underlies its important physiological contribution to Ca2+ dependent gene expression and cytokine release. 2.STIM2 proteins differentially modulate Orai1/STIM1 function: The recently identified STIM2 displays poor interaction with Orai1 and is reported to attenuate SOCE. Its Ca2+ sensitivity is similar to that of STIM2 and it also clusters with STIM1 and STIM2. Its SOAR domain has an additional sequence that is proposed to interfere with activation of Orai1. Our previous studies revealed that STIM2 can recruit Orai1 and STIM1 into ER-PM junctions and trigger a conformational change in STIM1. This results in STIM1-mediated Orai1 activation in cells with high ER-Ca2+. In this study, we have examined the effect of STIM2 on STIM1/Orai1 function. Our findings show that while STIM2 does not activate Orai1, it promotes STIM1-gating of Orai1. STIM2 when co-expressed withSTIM1, but not with STIM1-L394H or STIM1-R426L, caused constitutive activation of endogenous Orai1. Activation of endogenous Orai1, was increased in the following order: STIM2 < STIM2+STIM2 STIM2 < STIM2+STIM1 < STIM2+STIM1. STIM1 activates Orai1 only after store depletion. We suggest that the Orai1-mediated Ca2+ entry with STIM2+STIM1 is lower than that with STIM2+STIM1 due to the contribution of a relatively weaker gating of Orai1 by STIM2. Importantly, our findings show that although STIM2 and STIM2 exert differential effects on Orai1, both proteins recruit and trigger STIM1/Orai1 coupling. 3. Assessment of dynamic status of endogenous STIM/Orai proteins: Till date all studies reporting clustering patterns of the proteins within ER-PM junctions, have been done using overexpression systems utilizing tagged proteins. Most of these use strong promoter based expression systems. Thus the status of endogenous proteins and how they respond to store-depletion is not known. For example, the temporal characteristics of their mobilization, the size/nature of endogenous STIM clusters within ER-PM junctions for all three components of SOCE have not yet been studied. We have utilized CRISPR/Cas9 technique to knock-in a fluorescent tag (mVenus) into the endogenous STIM2-N terminus in HEK293 and HEK293T cells. We also use a weak (Thymidine kinase) promoter for protein expression and have standardized conditions to express protein at levels <2-fold over endogenous. We find that endogenous STIM2 localizes in the ER-PM junction (pre-clustered) under resting conditions in the cell. Analysis of TIRFM data reveal that majority of STIM2 at ER-PM junctions are unstable (mobile/dynamic) and that the number of stable STIM2 clusters (Immobile) increases after treatment with agonist. This report aims to elucidate the cellular factors that govern STIM2 pre-clustering. Immobile STIM2 clusters are the critical sites where STIM1 and Orai1 are recruited under low agonist stimulus. In the absence of Extended-Synaptotagmins (E-Syt2/3), ER-PM tethers, STIM2 does not appear to precluster under basal stimulus, although few dynamic or immobile clusters appears under TIRF plane. In such a condition, STIM2 does not cluster with STIM1/Orai1 in response to low CCh and substantial loss of SOCE was observed. We have further found an important link between the localization and function of IP3R and STIM2 pre-clustering. Low level expression of IP3R demonstrated presence of stable pre-clusters of the ER Ca2+ channel in ER-PM-junctions. In cells expressing IP3R and STIM2, we noted that STIM2 co-localized with stable IP3R. Use of IP3R-triple KO cell lines demonstrated that stable STIM2 pre-clusters are determined by the presence and localization of IP3R. cAMP can phosphorylate IP3Rs to enhance their function under ambient IP3 and Ca2+. Treatment of cells expressing WTIP3R1 with forskolin potentiated Stim2 clustering. STIM2 expression in cells with IP3RKO, or expressing mutant receptors (P-deficient and gating deficient) did not respond to forskolin. Finally, IP3R clustering was abolished by si ESyt2 and 3. Together our data elucidate novel characteristics of STIM2 and a critical role for IP3R in assembly of STIM/Orai1 complexes. The location of IP3R and STIM2 in ER-PM junctions, and the site of the latter, is most likely determined by the proximity to where receptor-mediated PIP2 hydrolysis occurs and IP3 is generated. IP3R can rapidly sense local IP3 and respond causing depletion of ER-Ca2+. STIM2 localized within the clusters or recruited after IP3R activation can sense local decrease in ER-Ca2+ and initiate the assembly of Orai1/STIm1 complex and activation Thus, ER-PM junctions where IP3R and STIM2 are localized are extremely relevant physiologically. Based on other findings from our lab, we can also propose that regulation of downstream Ca2+-dependent functions is also coordinated within these microdomains.